Identification of Enterococcus faecalis in Root-filled Teeth With or Without Periradicular Lesions by Culture- dependent and—Independent Approaches G. O. Zoletti, DDS,* J. F. Siqueira, Jr, DDS MSc, PhD, † and K. R. N. Santos, BS, MSc, PhD* Abstract Enterococcus faecalis is the most commonly found spe- cies in root-filled teeth evincing recalcitrant periradicu- lar lesions and as a consequence, a role in causation of endodontic treatment failure has been suggested. The purpose of this study was to evaluate the prevalence of this bacterial species in root-filled teeth with or without periradicular lesions. Identification of E. faecalis was carried out by polymerase chain reaction (PCR) or con- ventional culture procedures. Overall, E. faecalis was detected by species-specific 16S rRNA gene-based PCR in 40/50 teeth (80%), while culture revealed the occur- rence of this species in 8/50 teeth (16%). PCR was significantly more effective than culture in detecting this bacterial species (p  0.001). Of 27 root-filled teeth with no periradicular lesions, E. faecalis was found in 22 cases (81.5%) by PCR and in five cases (18.5%) by culture. Of 23 root-filled teeth with perira- dicular lesions, E. faecalis was identified in 18 cases (78%) by PCR and in three cases (13%) by culture. Regardless of the identification technique used, no significant difference was observed when comparing the occurrence of E. faecalis in root-filled teeth with and without periradicular lesions (p  0.05). Although these findings apparently put into question the status of E. faecalis as the main species causing endodontic treatment failure, other related factors still need to be clarified before this assumption turns into certainty. (J Endod 2006;32:722–726) Key Words Apical periodontitis, culture, endodontic retreatment, Enterococcus faecalis, PCR A large body of scientific evidence indicates that microorganisms are the major causative agents of endodontic failure characterized by persistence or appearance of a periradicular inflammatory lesion after treatment (1). Even though extraradicular infections can be responsible for some cases of failed endodontic treatment (2), per- sistent or secondary intraradicular infections have been the most common findings in cases selected for retreatment because of unsatisfactory outcome (3–8). Microbiological culturing techniques have been traditionally used to investigate the microbiota associated with endodontic infections and have revealed that Entero- coccus faecalis is the most frequently found species in persistent/secondary intrara- dicular infections associated with endodontic treatment failure (3–7). Recently, mo- lecular methods have been used to investigate the microbiota of endodontic infections, and the list of putative pathogens involved with failed endodontic therapy has expanded to include even as-yet-uncultivated bacteria (9, 10). However, in addition to detecting new putative pathogens, molecular biology studies have confirmed the status of E. faecalis as the most frequently found species in previously filled root canals that have failed (4, 5). Furthermore, molecular biology methods have strengthened the relation- ship between persistent/secondary intraradicular infections and endodontic treatment failure, since virtually all examined cases of root-filled teeth with persistent periradicu- lar diseases have been shown to harbor microorganisms (4, 5, 11). Because E. faecalis has been found in high prevalence in root-filled teeth with periradicular disease, it has been suggested that this species is involved in the pathogenesis of recalcitrant lesions. Nevertheless, there are some reports in the literature that have demonstrated that enterococci can also be found in root-filled teeth with no lesions. Engström (12) investigated the occurrence of enterococci in different types of endodontic infections and reported the isolation of these bacteria in 24% of the root-filled teeth with lesions and in 18% of the root-filled teeth with no lesions. Molander et al. (7) examined the microbiological conditions of root-filled teeth and isolated enterococci from 47% of the teeth with periradicular lesions and from 11% of the teeth without lesions. More re- cently, Kaufman et al. (8) detected enterococci in 6% of the root-filled teeth with lesions and in 23% of the root-filled teeth with no lesions. In addition, some studies have not succeeded in detecting enterococci in root-filled teeth with lesions (9, 13) or have demonstrated that E. faecalis is not the dominant species in retreatment cases (11). All of these reports question the involvement of E. faecalis in causing endodontic failures. Is this species actually participating in causation of persistent periradicular diseases or is this species only present in the root canal because of its ability to survive in bleak environments like a treated root canal? Based on these premises, the purpose of this study was to provide additional knowledge of the role of E. faecalis in treatment failures by investigating the occurrence of this species in cases of root-filled teeth with or without periradicular disease using two microbiological identification methods—16S rRNA gene-based PCR and culture. The study design also allowed the comparison of the efficacy of these two methods in detecting E. faecalis in root-filled teeth. Materials and Methods Case Description Samples were collected from 53 root-filled teeth from consecutive adult patients (ages ranging from 19 to 75 years) who had been referred to the Endodontic Clinic at From the *Department of Microbiology, Institute of Mi- crobiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; † Department of Endodontics, Faculty of Dentistry, Es- tácio de Sá University, Rio de Janeiro, Brazil. Address requests for reprints to José F. Siqueira, Jr., Av. Almte Ary Parreiras 311/1001, Icaraí, Niterói, RJ, Brazil 24230- 322. E-mail address: jfsiqueira@yahoo.com. 0099-2399/$0 - see front matter Copyright © 2006 by the American Association of Endodontists. doi:10.1016/j.joen.2006.02.001 Clinical Research 72 2 Zoletti et al. JOE — Volume 32, Number 8, August 2006